1. Field of the Invention
The present invention relates to a resin and method for the specific removal of plasmin(ogen) and its derivatives from protein solutions, where the resulting protein solution can be used for intravenous administration and for local applications, i.e. matrix support for sustained release and healing of wounds, either as a sole active component or combined with other pharmaceutical, acceptable drugs. The removal of plasmin(ogen) would preserve the integrity and the function of the protein solution for longer incubation periods. This invention is also related to the production of highly purified plasmin(ogen) for therapeutic use.
2. Related Art
Plasminogen or its active molecule plasmin (in the following plasmin(ogen)), very frequently contaminates protein solutions, especially those extracted from animal fluids or animal organs. The presence of plasmin(ogen) in a protein solution presents a multiple threat to its acceptance as a stable pharmaceutical product, due to the molecule's known proteolytic activity on various protein and peptides at arginyl and lysyl peptide bonds (Weinstein M. J., Doolittle R F. Differential specificities of the thrombin, plasmin and trypsin with regard to synthetic and natural substrates and inhibitors RF Biochim Biopliys Acta. 1972 258:577-90 and Ling C M, Summaria L, Robbins K C. Mechanism of formation of bovine plasmin(ogen) activator from human plasmin. J Biol chem. 1965. 240:4212-B); and basic amino acids, its stimulatory activity on various tissues, especially the central nerve tissue and its role in binding (Chen Z L, Strickland S Neuronal death in the hippocampus is promoted by plasmincatalyzed degradation of liminin. Cell. 1997. 91:917-25) and probably carrying prions in the blood of mammals Fischer (M B, Roeckl C, Parizek P, Schwarz H P. Aguzzi A Binding of disease associated prion protein to plasmin(ogen). Nature. 2000. 408:479-83).
Several chromatographic methods were developed for the purification of plasmin(ogen) from protein solutions and hence, removing plasmin(ogen) from protein solution.
These methods are essentially based on two principles. The first group is based on several consecutive purification steps that utilize the differential solubility, isoelectric point, or molecular size distribution Alkjaerisig N. (The purification and properties of human plasmin(ogen). Biochem. J. 1963, 93:171-182). Since their prime target was to purify plasmin(ogen), these methods totally distorted the composition of the protein solution. The second group of methods is based on one step affinity purification. The purification is based on binding plasmin(ogen) to various synthetic ω-amino carboxylic acid ligands that can bind onto the lysine binding sites on the plasmin(ogen) heavy chain. These sites, consist of 5 triple loop disulfide bridges with internal sequence homology known as the plasmin(ogen) kringles, located on the NH2 plasmin(ogen) heavy chain and far from the catalytic site located on the COOH light chain, bind fibrin(ogen). Another possibility for affinity chromatography is to bind plasmin(ogen) via the catalytic site, a potentially less specific binding since it may bind many proteins such as serine proteases having similar or lower affinity to arginyl and lysyl peptide bonds and basic amino acids. In summary, it might be concluded that in general, plasmin(ogen) affinity chromatography is performed by a given ligand that chemically and ionically resembles ω-amino-carboxylic acid or the substrate of the plasmin catalytic site. The ligand is bound to the resin through an adequate spacer or linker. However an ideal affinity resin for the removal of plasmin(ogen) is not essentially the same resin found ideal for the purification of plasmin(ogen). Such resins should contain a ligand that binds plasmin(ogen) at high affinity and has very low affinity to other proteins such as other serine proteases and especially very low affinity for fibrinogen which is the main protein in Plasma Cohn's fraction I or in cryoprecipitate. It is also important that the removal of plasmin(ogen) by using the given affinity chromatography might be performed in a wide range of buffers and not be restricted to a certain buffer that may endanger the stability and the integrity of proteins in the solution, those being a main concern and not the plasmin(ogen).
The antifibrinolytic potency (ability to inhibit the binding of plasmin(ogen) to fibrinogen at high affinity) of the -amino-carboxylic acids depends on the presence of free amino and carboxylic group and on the distance between the COOH-group and the carbon atoms to which the NH2-group is attached (Markwardt 1978) such as -amino caproic acid (EACA), and p-amino benzamidine (PAMBA). Comparison between the antifibrinolytic activities of EACA and PAMBA showed that the latter is about three times more active. Shimura et al (1984) designed a resin in which p-amino benzamidine was bound to microparticles of hydrophilic vinyl polymer via a spacer (linker) moiety. By using this resin, Shimura et al were able to separate plasmin and plasminogen by high performance affinity chromatography. The facts that plasmin(ogen) could not be eluted by 6-aminohexanoic acid alone and that 3 M urea had to be included in the elution buffer indicated a two-site interaction of plasmin with this immobilized ligand i.e., the lysine-binding sites on the heavy chain and the catalytic site on the light chain. This may explain the finding by other researchers that p-amino benzamidine removes also some other proteins.
Another resin, the lysine-resin, is manufactured and used for the affinity purification of plasmin(ogen). However, the antifibrinolytic potency of lysine is very low and thus, also its binding affinity. It also binds to other proteins and its specificity is buffer dependent.
Moroz L A. Gilmore N J Fibrinolysis in normal plasma and blood: evidence for significant mechanisms independent of the plasminogen-plasmin system, Blood, 1976, 48, 531-45 disclose a preparation of plasmin(ogen)-free plasma by affinity chromatography. Based on the methods employed the authors report about observations indicating that processes which culminate in the generation of the fibrinolytic enzyme plasmin play at most a minor role in the spontaneous or basal fibrinolytic activity measurable in normal human plasma. Tranexamic acid was used together with other protease inhibitors as plasmin inhibitors for measuring fibrinolytic activity. For preparation of the plasmin(ogen)-free plasma the method of Deutsch and Meltz, Science 170; 1095-1096, 1997 was employed.
Iwamoto in Thrombos. Diathes. Heamorrh. (Stuttg.), 1975, 33, 573 discloses specific binding of a tranexamic acid to plasmin. Although tranexamic acid is identified as a powerful ligand of plasmin, it is indicated that the anti-fibrinolytic effect of tranexamic acid is a result of not only the binding to plasmin(ogen), but also of the enhancement of cooperation of the natural antiplasmins. Therefore one would conclude that binding of tranexamic acid to a solid support will not only remove plasmin(ogen) from plasma but also the natural antiplasmins. One would also suggest that tranexamic acids may cause of formation of aggregates (conglomerates) with plasmin inhibitors. This understanding is based on the discrepancy, which can be found when comparing the anti-fibrinolytic activity of ε-amino-caproic acid and tranexamic acid resulting in 98 and 91% inhibition in urokinase-stimulated plasma versus plasma that has been heparinized oral blood (65 and 39% for ε-amino-caproic acid and tranexamic acid respectively—cf. tables 2 and 7 in the Moroz et al. paper). One would expect that due to their high binding ratio tranexamic acid and ε-amino-caproic acid are good candidates for high affinity ligands. However, one would also expect this ligands would block an affinity column as a result binding of plasmin and plasmin inhibitor complexes.